The present invention relates generally to chemical compounds and compositions and their use as immunomodulatory agents.
Copending applications, Ser. No. 536,993 and Ser. No. 536,994, disclose the formation of novel compounds, one use for which is also disclosed herein. These compounds are produced by reacting approximately equal amounts of compound of the general formula ##STR1## wherein R.sub.1 and R.sub.2 are selected from the group consisting of hydrogen and lower alkyl, provided that at least one is hydrogen; wherein R.sub.3 and R.sub.4 may be the same or different and are selected from the group consisting of lower alkyl and aryl; wherein R.sub.5 and R.sub.6 are selected from the group consisting of hydrogen and lower alkyl and may be the same or different and a compound of the general formula ##STR2## wherein X is selected from the group consisting of O, S, and NH; wherein R.sub.7 and R.sub.8 are selected from the group consisting of hydrogen and lower alkyl and may be the same or different. wherein R.sub.7 may be ##STR3## wherein R.sub.9 is selected from the group consisting of ##STR4## wherein R.sub.10 and R.sub.11 are selected from the group consisting of hydrogen, lower alkyl, phenyl and hydroxy substituted lower alkyl and may be the same or different obtaining the product of the reaction, treating the product with acid anhydride or acid amide, refluxing and crystallizing the novel compounds. Recovery of the pure product may be facilitated by treatment or purification of the intermediate by any of the several techniques known to one of ordinary skill in the art such as, for example, charcoal filtration, ion exchange or gel chromotography.
The novel compounds produced by the above described process include ##STR5## wherein R.sub.2 is selected from the group consisting of hydrogen and lower alkyl; wherein R.sub.5 and R.sub.6 are selected from the group consisting of hydrogen and lower alkyl and may be the same or different; wherein R.sub.7 and R.sub.8 are selected from the group consisting of hydrogen and lower alkyl and may be the same or different; wherein R.sub.7 may be ##STR6## wherein R.sub.9 is selected from the group consisting of ##STR7## wherein R.sub.10 and R.sub.11 are selected from the group consisting of hydrogen, lower alkyl, phenyl and hydroxy substituted lower alkyl and may be the same or different; wherein when R.sub.7 contains a hydroxyl group in the .alpha., .beta., or .gamma. position, R.sub.7 may form the hemiketal ring closure at carbon 3 of the butyrolactone with protonation of the carbonyl group on the same carbon atom; wherein n is selected from the group consisting of 1, 2, and 3; and
wherein X is selected from the group consisting of O, S, and NH.
Lactone ring formation, ##STR8## wherein R.sub.2, R.sub.5, R.sub.6, R.sub.10, X and n are as described above can occur. These rings will ordinarily partially rearrange, in aqueous or highly polar media to give tautomeric equilibrium products ##STR9## wherein R.sub.2, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.10, R.sub.11, X and n are as described above.
The compounds disclosed in Ser. No. 1 and Ser. No. 2 can be formulated with generally known pharmaceutical carriers into compositions which can be administered to animals and humans. These compositions show immunomodulatory and cytotoxic activity at very low accute toxicity values.
It is believed that the cytotoxic activity of these compositions may be attributable to stimulation of the immune system by the compounds or to the cytoxicity of the compounds per se or to a combination of both effects.
Because of their cytotoxic activity, these compositions may be used in cancer therapy for a broad range of cancers. The compositions may be used in immune therapy of depressed immune systems, as in the case of AIDS (Acquired Immune Disorder Syndrome).
The immune system is one of the primary defenses against disease bearing microbes and other foreign protein in higher animals. An immune response is mediated by the action of specific antibody proteins which react to specific antigens. Antigens are substances of fairly high molecular weight, often proteins, which are foreign to an individual's body. They are most frequently located on the outer surfaces of cells. Potential antigens can be found on pollen grains, tissue grafts, synthetics, animal parasites, viruses, and bacteria.
In humans, many potential antigens never pass the body's first two defense lines and therefore never trigger the immune system. These two defense lines consist firstly of the skin, mucous membranes, tears, and stomach acid and secondly of specialized white blood cells, granulocytes and monocytes, and macrophages which destroy pathogens and other potential antigens by phagocytosis, that is by engulfing and destroying the foreign material. These white blood cells and macrophages are called phagocytes. When pathogens or other foreign substances do pass the body's first two defense lines, the immune response begins.
There are two principal immune defense systems, humoral and cellular, both of which react to antigens. Humoral immunity is due to circulating antibodies which are found in the gamma globulin fraction of the plasma proteins. When plasma is centrifuged at high speeds its component proteins separate by weight into sections called fractions. Antibodies are usually found in the fraction whose components have a molecular weight of approximately 156,000. This particular fraction has been named the gamma globulin fraction. Humoral immunity forms a major defense against bacterial infections. Cellular immunity is partly due to lymphocyte products called lymphokines. This type of immunity is responsible for delayed allergic reactions, rejection of transplants of foreign tissue, and rejection of tumor cells. It is the major defense against infections due to viruses, fungi, and a few bacteria such as the tubercle bacillus.
Specialized white blood cells called lymphocytes are responsible for both humoral and cellular immunity. Lymphocyte precursor cells are made in the bone marrow of adult humans followed by migration to various organs or in the yolk sac of a developing fetus followed by migration into the fetus and then to various organs. In humans, some of these precursor cells migrate to the thymus, which is a two-lobed, glandular appearing structure located in the upper chest just behind the sternum, where they are transformed into T-lymphocytes which are involved in cellular immunity. In humans, the rest of the precursor cells migrate to the spleen where they are transformed into B-lymphocytes which are involved in humoral immunity. The T- and B-lymphocytes are structurally indistinguishable although they function differently and can be distinguished through various chemical means. The mature lymphocytes circulate in the blood and can also be found in the lymph nodes as well as the spleen and thymus.
Humoral immunity is mediated by the B-lymphocytes which have receptors for particular antigens on their cell surfaces. They seem to be very specific and each type of B-lymphocyte reacts to only one antigen. When bacteria or viruses, for example, invade an organism, B-lymphocytes react to and combine with the antigens on the bacterial or viral surface and the lymphocyte is stimulated to divide. Its daughter cells are transformed into specialized cells called plasma cells. These cells produce and then secrete large quantities of antibodies into the general circulation. The antibodies are specific for the antigens which stimulated their production and react only with those antigens. Antibodies known as agglutinins cause several antigen containing substances to agglutinate or clump together. This keeps the substance from spreading to the tissues and allows the phagocytes to capture or the lymph nodes to filter the invading material. Other antibodies act by opening holes in bacterial cell walls, thereby killing the bacteria. These are known as lysins. Antibodies called antitoxins combine with toxins produced by bacteria and thereby neutralize them.
Once a pathogen invades the body and the immune response begins, antibodies can be made in several hours. This initial reaction is called the primary response or primary immunization. However, during that time, the pathogens have also been dividing and sometimes producing toxin, either of which results in various disease symptons. It may take days or weeks before enough antibodies are made to eliminate all the pathogens but one they disappear, the disease symptoms disappear as well. The lymphocytes, plasma cells, and antibodies remain and circulate in the blood so that if the same pathogens enter the body a second time, the lymphocytes react immediately and start antibody production. The response of the sensitized lymphocytes is called the secondary response. The secondary response results in the production of higher levels of antibody than were produced during the primary response. So many antibodies are produced so rapidly that the microbes are unable to divide and cause disease. Humoral immunity is known as immediate hypersensitivity due to the fact that a previously exposed organism can respond within minutes to an antigen, as in the case of hay fever. Another example of immediate hypersensitivity would be anaphylactic shock, an extreme allergic reaction that sometimes occurs when an individual is exposed to an antigen to which he has been sensitized. At times, this humoral response to the antigen can result in death.
Humoral immunity can be both naturally and artificially induced. In the case of active natural immunity, an individual's lymphocytes continue to circulate and activate the production of antibodies after an infection. This active natural immunity lasts for many years or even a lifetime. An infant receives antibodies from the colostrum, milk secreted by the mother, the first few days after birth, which gives it immunity the first year of its life. This is known as passive natural immunity since the infanct is not involved in the actual production of the antibodies. Active artificial immunity is induced by injecting dead or weakened microbes into an individual. Their surface antigens can still trigger lymphocyte production of antibodies but these microbes do not cause the disease symptoms that their more virulent forms do. When the individual is later exposed to the virulent microbe, he is already sensitized and immediately responds with a massive production of antibodies. Active artificial immunity may last many years or permanently with booster shots. There is also a form of passive artificial immunity which provides protection for about one month. This temporary immunity is brought about by injecting antibodies obtained from another person or animal into an individual. It is usually only used in crisis situations and epidemics. Because the lymphocytes are bypassed, they neither make antibodies nor "remember" the antigen, which accounts for the temporary effect of this method.
In cellular immunity, as contrasted to humoral immunity, circulating antibodies are not detectable. The T-lymphocytes which mediate this type of immunity are activated when they encounter antigens on cells from another individual, as in the case of transplants, tumors, or viruses. Like B-lymphocytes, T-lymphocytes are specific and each type reacts with only one antigen. The lymphocytes enlarge, divide, and produce lymphokines which participate in the attack on the foreign protein. They also stimulate the phagocytic activity of macrophages. Although immunological memory exists as with humoral immunity, the response is much slower. It may take as long as ten or twelve hours to develop a response in a previously sensitized individual and cellular immunity is therefore known as delayed hypersensitivity. The allergic reaction to poison ivy, oak, and sumac, the red splotch seen in a positive tuberculin skin test, and rejection of transplant tissue are all cellular immune responses.
Immunomodulating agents activate or inhibit the process of lymphocyte proliferation. Normal lymphocyte proliferation is due to various interactions between antigens, macrophages, T- and B-lymphocytes as well as certain chemicals. For example, the presence of a particular antigen activates a particular T- or B-lymphocyte. Additionally, certain B-lymphocytes can be activated by active T-lymphocytes while others are independent of the T-lymphocytes and are activated only by antigens. Activated T-lymphocytes can cause macrophages to produce a molecule known as interleukin 1(IL-1) which inturn activates both T- and B-lymphocytes. Activated T-lymphocytes can also produce a molecule known as interleukin 2(IL-2) which further induces T-lymphocyte activation. Chemicals, called mitogens can trigger DNA synthesis and mitosis, which are signs of activity in T- or B-lymphocytes. Some mitogens affect only one type of lymphocyte while others affect many types. Immunomodulating agents of various kinds and in varying amounts affect the complex interactions between the components of the immune system. As will be shown, the compounds and compositions of this invention act as immune stimulators or suppressors, depending upon the dosage, and affect both T- and B-lymphocytes.
Although the immune system is a major defense against substances which can cause disease, it cannot distinguish between helpful and harmful foreign substances and destroys both. It would be useful in many instances to have a means of regulating the immune system without harming the individual. The compounds and compositions of this invention exhibit such modulating or regulatory effects and have potential for use in the treatment of various immune disorders.
The immune system has been linked to some aspects of aging and may be important in protecting against cancer. The system is necessary for the recognition of changing or aging cells, such as worn out red blood cells, and their subsequent destruction, and for this reason is vital to normal body functions. One theory in the case of cancer is that the transformation of cells to the malignant state may occur fairly frequently but these changed cells are recognized as "not self" and destroyed. Some carcinogens may work by depressing the immune response rather than by transforming cells themselves to a malignant state. This would mean that the body would no longer destroy the naturally transformed cells and a cancerous growth could result. Immunostimulation could be useful in treating cancers of this type. When used as adjunctive therapeutic agents in chemo- or chemoimmuno-therapy, the compounds and compositions of this invention can affect the growth of tumors.
Some of the methods of treating cancer, surgery, chemotherapy, and radiation for example, can result in a suppression or drastic variation of the normal functions of the immune system. Immunostimulatory drugs, such as the compounds and compositions of this invention, can be very effective in combating and/or preventing various infections which can result due to the depressed immune system. Some immunomodulating agents which are currently being studied for use in these situations are cyclosporin-A, levamisole, and isoprinosine (inosiplex). The compounds and compositions of this invention will be novel therapeutic tools for use in this and other situations involving the immune system.
There are times when the individual's immunological response causes more damage or discomfort than the invading microbes or foreign material, as in the case of allergic reactions. Suppression of the immune response in these cases would be desirable.
Occasionally, the immunological mechanisms become sensitized to some part of the individual's own body causing interference with or even destruction of that part. The ability to distinguish between "self" and "not self" is impaired and the body begins to destroy itself. Some examples of these autoimmune diseases in man are rheumatoid arthritis, certain hemolytic anemias, rheumatic fever, thyroiditis, ulceractive colitis, myesthenia gravis, glomerulonephritis--a kidney disease, allergic encephalomyelitis, continuing nerve and liver destruction which sometimes follows viral hepatitis, and possibly multiple sclerosis. Some forms of autoimmunity come about as the result of trauma to an area usually not exposed to lymphocytes such as neural tissue or the lens of the eye. When the tissues in these areas become exposed to lymphocytes, their surface proteins can act as antigens and trigger the production of antibodies and cellular immune responses which then begin to destroy those tissues. Other autoimmune diseases develop after exposure of the individual to antigens which are antigenically similar to, that is cross-react with, the individual's own tissue. Rheumatic fever is an example of this type of disease in which the antigen of the streptococcal bacterium which causes rheumatic fever is cross-reactive with parts of the human heart. The antibodies cannot differentiate between the bacterial antigens and the heart muscle antigens and cells with either of those antigens can be destroyed. Suppression of the immune system in these autoimmune diseases could be useful in minimizing or eliminating the effects of the disease.
Circulating antibodies and cellular immune responses play a role in the rejection of transplanted tissues and organs. Unless the donor is the identical twin of the recipient or is the individual himself, the recipient's lymphocytes recognize the transplant as "not self" and immediately respond to destroy it. The exceptions to this situation are transplants to non-vascularized areas (privileged sites), such as the cornea of the eye, where lymphocytes do not circulate and therefore are not sensitized and do not prompt an immune response. It is currently difficult to suppress the immune reaction to prevent rejection of the transplant without severly damaging the patient in other ways. The patient must also be given massive doses of antibiotics because his own defenses against infection have been suppressed. The compounds and compositions of this invention could be valuable in establishing tolerance to the transplant through controlled modulation of the immune system.
Because of the immune system's vital roles in defense against disease and in normal body functions, compounds and compositions, such as those disclosed in this invention, which can enhance or diminish that role are of importance. They are particularly useful when the normal action of the system has been disturbed or has caused the destruction of harmless foreign materials. They can also assist by stimulating the system's response to harmful antigens and, thus, shorten the course of various diseases.